The present invention relates generally to methods of treating gram-negative bacterial infections and the sequelae thereof and more specifically to the use of bactericidal/permeability-increasing protein (BPI) and BPI protein products in treatment of such infections.
BPI is a protein isolated from the granules of mammalian polymorphonuclear leukocytes (PMNs), which are blood cells essential in the defense against invading microorganisms.
Human BPI protein has been isolated from polymorphonuclear neutrophils by acid extraction combined with either ion exchange chromatography [Elsbach. J. Biol. Chem., 254:11000 (1979)] or E. coli affinity chromatography [Weiss et al., Blood, 69:652 (1987)] referred to herein as natural BPI and has potent bactericidal activity against a broad spectrum of gram-negative bacteria. The molecular weight of human BPI is approximately 55,000 daltons (55 kD). The amino acid sequence of the entire human BPI protein, as well as the DNA encoding the protein, have been elucidated in FIG. 1 of Gray et al., J. Biol. Chem., 264:9505 (1989), incorporated herein by reference.
The bactericidal effect of BPI has been shown to be highly specific to sensitive gram-negative species, while being non-toxic for other microorganisms and for eukaryotic cells. The precise mechanism by which BPI kills bacteria is as yet unknown, but it is known that BPI must first attach to the surface of susceptible gram-negative bacteria. This initial binding of BPI to the bacteria involves electrostatic and hydrophobic interactions between the basic BPI protein and the negatively charged sites on lipopolysaccharides (LPS). LPS has been referred to as "endotoxin" because of the potent inflammatory response that it stimulates. LPS induces the release of mediators by host inflammatory cells which may ultimately result in irreversible endotoxic shock. BPI binds to lipid A, the most toxic and most biologically active component of LPS.
In susceptible bacteria, BPI binding is thought to disrupt LPS structure, leading to activation of bacterial enzymes that degrade phospholipids and peptidoglycans, altering the permeability of the cell's outer membrane, and initiating events that ultimately lead to cell death. Elsbach and Weiss, Inflammation: Basic Principles and Clinical Correlates, eds. Gallin et al., Chapter 30, Review Press. Ltd. (1992). BPI is thought to act in two stages. The first is a sublethal stage that is characterized by immediate growth arrest, permeabilization of the outer membrane and selective activation of bacterial enzymes that hydrolyze phospholipids and peptidoglycan. Bacteria at this stage can be rescued by plating on serum albumin supplemented media. The second stage, defined by growth inhibition that cannot be reversed by serum albumin, occurs after prolonged exposure of the bacteria to BPI and is characterized by extensive physiologic and structural changes, including penetration of the cytoplasmic membrane.
BPI is also capable of neutralizing the endotoxic properties of LPS to which it binds. Because of its gram negative bactericidal properties and its ability to neutralize LPS, BPI can be utilized for the treatment of mammals suffering from diseases caused by Gram-negative bacteria, such as bacteremia or sepsis.
A proteolytic fragment corresponding to the N-terminal portion of human BPI holoprotein possesses substantially all the antibacterial efficacy of the naturally-derived 55 kD human holoprotein. In contrast to the N-terminal portion, the C-terminal region of the isolated human BPI protein displays only slightly detectable anti-bacterial activity. Ooi et al., J. Exp. Med., 174:649 (1991). A BPI N-terminal fragment, which is the expression product of a gene encoding the first 199 amino acid residues of the human BPI holoprotein and comprising approximately the first 193 to 199 amino acid residues of human BPI hgoloprotein and referred to as "rBPI.sub.23 ", has been produced by recombinant means as a 23 kD protein. Gazzano-Santoro et al., Infect. Immun. 60:4754-4761 (1992).
Lipopolysaccharide binding protein (LBP) is a 60 kD glycoprotein synthesized in the liver which shows significant structural homology with BPI. LBP is found in the serum of normal humans at levels of 5-10 .mu.g/mL but can reach levels of 50-100 .mu.g/mL in septic patients. Schumann et al., Science, 249:1429 (1990) disclose the amino acid sequences and encoding cDNA of both human and rabbit LBP. Like BPI. LBP has a binding site for lipid A and binds to the LPS from rough (R-) and smooth (S-) form bacteria. Unlike BPI. LBP does not possess significant bactericidal activity. BPI has been observed to neutralize and inhibit LPS-induced TNF production resulting from interaction of LBP with CD14 on monocytes and macrophages. Marra et al., J. Immunol. 148: 532 (1992), Weiss et al., J. Clin. Invest. 90: 1122 (1992). In contrast, LBP is observed to enhance LPS-induced TNF production. Wright et al., Science, 249:1131 (1990). Thus, in contrast to BPI, LBP has been recognized as an immunostimulatory molecule. See, e.g., Seilhamer, PCT International Application WO 93/06228 which discloses a variant form of LBP which it terms LBP-.beta..
Recently, it has been discovered that there exist biologically active protein derivatives of LBP which are characterized by the ability to bind to LPS but which lack CD14-mediated immunostimulatory properties of the LBP holoprotein. Specifically, co-owned and copending U.S. patent application Ser. No. 08/261,660 (Gazzano-Santoro et al., "Lipopolysaccharide Binding Protein Derivatives") filed Jun. 17, 1994, now U.S. Pat. No. 5,731,415, which is a continuation-in-part of U.S. patent application Ser. No. 08/079,510 filed Jun. 17, 1993, now abandoned, the disclosures of which are hereby incorporated by reference, discloses LBP protein derivatives and LBP derivative hybrid proteins which are capable of binding LPS and which lack CD14-mediated immunostimulatory activity. Preferred LBP protein derivatives have been produced by recombinant expression of genes encoding amino terminal amino acid residues, such as amino acid residues 1-197, and the resulting protein designated rLBP.sub.25.
Of interest to the present application are the disclosures of references which relate to the potentiation of BPI bactericidal activity by 15 kD proteins derived from the granules of rabbit PMNs designated p15. Ooi et al. J. Biol. Chem., 265:15956 (1990) disclose two related 15 kD proteins derived from rabbit PMN granules which have no bactericidal activity by themselves but which potentiate the first sublethal stage of BPI antibacterial activity but have an inhibitory effect on the second lethal stage of BPI antibacterial activity. Levy et al., J. Biol. Chem., 268: 6058-6063 (1993) disclose the sequences of cDNAs encoding the two rabbit proteins and report that the protein with the stronger potentiating effect reduces the required dose of BPI for the early bacteriostatic effect by about 20-fold.